One family that has an abnormal von Willebrand factor (vWf) with a defective binding site for factor VIII has been studied, and the genetic defect has been identified. The binding defect was initially evaluated by assessing the ability of the patient's vWf to bind purified factor VIII. Subsequently, DNA was purified from peripheral blood leukocytes, specific regions of the vWf gene were amplified by polymerase chain reaction (PCR), and direct sequencing of the DNA was carried out. A transition of nucleotide 2451 (T to A) was found, which results in the substitution of GLN for HIS at amino acid 54 in the mature vWf subunit. We recently used a PCR mutagenesis technique to insert the mutation into cloned DNA and have expressed the abnormal protein. The latter was tested in an assay for binding factor VIII and was shown to manifest decreased binding of factor VIII.An unrelated patient with von Willebrand's disease and an abnormal distribution of vWf multimers has been studied, and a new mutation in the A1 region of the vWf gene has been identified. The mutation was cloned into an expression vector and the expressed abnormal vWf is being characterized.